Bacteria and most other unicellular organisms possess a cell wall, which comprises a cross-linked polysaccharide-peptide complex called peptidoglycan. Peptidoglycan biosynthesis consists of three stages: (1) synthesis of precursors (sugar nucleotides) in the cytosol, (2) precursor transfer across the membrane and formation of the polysaccharide chain, and (3) cross-linking of individual peptidoglycan strands in the cell wall.
In the latter stage of peptidoglycan biosynthesis, new bonds must be made between nascent glycan strands and existing peptidoglycan. The newly synthesized chains are about 10 disaccharides long and are extended by transglycosylase enzymes to a final glycan strand of between 100 and 150 disaccharide units. The peptidoglycan is crosslinked by the action of transpeptidases which link the terminal D-ala of one glycan strand to a free .epsilon.-amino group on a diaminopimelic acid residue on an adjacent region.
A number of antibiotics inhibit bacterial growth by interfering with the formation of the peptidoglycan layer. The cross-linking reaction is the target for action of two important classes of such antibiotics, the penicillins and the cephalosporins. Penicillin is thought to react irreversibly with the transpeptidase that catalyses cross-linking.
The penicillin interactive proteins fall into three groups: the .beta.-lactamases, the Low Molecular Weight-Penicillin Binding Proteins (PBPs), which mainly include the carboxypeptidases, and the High Molecular Weight-Penicillin Binding Proteins. Penicillin Binding Proteins are those enzymes which have been shown to bind radiolabelled penicillin G. In Escherichia coli such proteins are called e.g. PBP 1A and PBP 1B, both belonging to the class High Molecular Weight-PBPs. PBP 1A and 1B, which are known to be membrane bound proteins, maintain cell integrity and control peptidoglycan side wall extension during growth. Inactivation of either PBP 1A or PBP 1B can be tolerated by the bacteria while the deletion of both the genes, designated ponA and ponB, is lethal (Yousif et al., 1985).
PBP 1B is known to be a bifunctional enzyme possessing both transpeptidase and transglycosylase activity (Ishino et al., 1980). PBP 1A is believed to be bifunctional since it can substitute for PBP 1B. The .beta.-lactam antibiotics, such as penicillin, inhibit only the transpeptidase activity of these proteins.
The transglycosylase reaction is inhibited by e.g. moenonmycin, which is a phosphoglycolipid used as a growth promoter in animal nutrition and which has been shown to possess broad spectrum bactericidal activity. The enzyme transglycosylase has been shown to be present in Escherichia coli, Staphylococcus aureiis, Bacillus megateriium and Bacillus subtilis. This suggests that interference of peptidoglycan biosynthesis by inhibition of transglycosylase could be a lethal event in all clinically important pathogens.
The putative transglycosylase domain of PBP 1B has been assigned to the N-terminal 478 amino acids (Nakagawa et al., 1987). This regions includes three conserved stretches of amino acids between the N-terminal half of both PBP 1A and 1B and could represent residues involved in the transglycosylase activity.
Preparation of Penicillin Binding Protein 2A from Staphylococcus aureus is disclosed in EP-A-0505151.